Breast cancer stem cells (BCSCs), as a subset of cancer cells with enriched capacity to generate breast tumors, have recently been attributed to driving cancer recurrence and metastasis. Although the importance of developing strategies to target BCSCs has been highlighted, it is conceivable that the depletion of BCSCs within a breast tumor would not lead to complete regression since non-BCSCs might still be capable of sustaining tumor growth or regaining BCSC potential. As either of these possibilities would confound the effectiveness of therapeutic agents that exclusively target BCSCs, this study aimed to develop combinatory therapeutics that apply agents targeting both BCSCs and non-BCSCs. By utilising parental cells and mammospheres as screening platforms, a total of 193 candidate molecules were identified as potent hit compounds inhibiting both BCSCs and non-BCSCs. Subsequent analysis suggested histone deacetylase (HDAC) inhibitors as a class of anti-cancer agents targeting both BCSCs and non-BCSCs. When combined with conventional chemotherapeutics, HDAC inhibitors were found to synergise DNA-damaging chemotherapeutics, namely doxorubicin and cisplatin, against BCSCs and non-BCSCs derived from MDA-MB- 468; whereas their combined treatment with anti-mitotic chemotherapy, namely paclitaxel, only exerted synergy in MDA-MB-468 non-BCSCs. Intriguingly, neither pan-HDAC, hydroxamate-based inhibitor nor class I specific, benzamide-based HDAC inhibitor was superior to be combined with conventional chemotherapeutics against these BCSCs and non-BCSCs. Further evaluations indicated that quisinostat, as the most potent HDAC inhibitor tested, managed to enhance the doxorubicininduced cytotoxicity in both BCSCs and non-BCSCs derived from different subtypes, including basal-like triple negative breast cancer (TNBC) subtypes (MDA-MB-468 and HCC38), mesenchymal-like TNBC (MDA-MB-231), and luminal-like breast cancer (MCF-7). Conversely, the synergism between quisinostat and cisplatin was TNBC subtype-dependent. Also, it was observed that both BCSCs and non-BCSCs of TNBC subtypes were more sensitive to the co-treatment of quisinostat with DNA damaging chemotherapeutics, as compared to luminal-like breast cancer subtype. Moreover, the dose reduction potentials of these combinatory regimens that revealed in this study may be exploited to reduce the dose-limited toxicities in clinical oncology. In summary, this study offered HDAC inhibitors as novel therapeutic options, either as monotherapy or combination therapy, for refractory breast cancer treatment which warrants detailed investigations.

Cancer stem cells (CSCs) are a subpopulation of cancer cells with enhanced capabilities of self-renewal, differentiation and tumour initiation, and confer resistance to conventional chemotherapy. Therefore, it is important to elucidate the signalling and regulatory mechanisms in breast CSCs, which will be of useful for the development of effective CSC-targeted therapy. Sphingosine kinases (SPHKs) are conserved lipid enzymes that catalyse the formation of sphingosine-1-phosphate (S1P) through ATP-dependent phosphorylation of sphingosine. Although compelling evidence has indicated the oncogenic roles of SPHK1 in breast cancer, the functions of SPHK1 and its regulatory mechanisms in breast CSCs remain poorly understood. Accordingly, the study presented herein sought to investigate the functions of SPHK1 and its regulatory mechanism in human breast CSCs, as well as whether these would be different in non-stem breast cancer cells. This study first described the hyperactivation of SPHK1/S1P axis in breast CSCs as compared to non-stem breast cancer cells. By utilising mammosphere culture and RNA-interference (RNAi) approaches, this study identified the functional roles of SPHK1 in mediating the survival and proliferation of both non-stem breast cancer cells and breast CSCs derived from HCC38 and MDA-MB-468, of which knockdown of SPHK1 significantly inhibited cell proliferation and induced apoptosis in all tested non-stem breast cancer cells and breast CSCs, while ectopic expression of SPHK1 promoted the survival and mammosphere forming efficiency of breast CSCs. Subsequently, this study discovered interferon (IFN)/STAT1 signalling as key targets of SPHK1 through global proteomic analysis and validated a novel mechanism by which SPHK1 promotes the survival of both non-stem breast cancer cells and breast CSCs via STAT1 suppression. Depletion of SPHK1 was found to induce apoptosis in both non-stem breast cancer cells and breast CSCs in a STAT1-dependent mechanism, along with activation of type I and II IFN signalling. Furthermore, this study reported that SPHK1 inhibitors including FTY720 and PF-543 synergised doxorubicin sensitivity in targeting both non-stem breast cancer cells and breast CSCs. In conclusion, this study identified SPHK1 as a key regulator of cell survival and proliferation in both non-stem breast cancer cells and breast CSCs, which warrants further development for theragnostic purposes in breast cancer treatment.